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AVANCES EN NUEVOS MARCADORES DE DAÑO RENAL EN EL DIAGNÓSTICO Y TRATAMIENTO DE LA
LEISHMANIOSIS CANINA
Samantha Guerrero Cabrera
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3 AVANCES EN NUEVOS MARCADORES DE DAÑO RENAL
EN EL DIAGNÓSTICO Y TRATAMIENTO DE LA LEISHMANIOSIS CANINA
Samantha Guerrero Cabrera
Directores:
Josep Pastor Milán Asta Tvarijonaviciute
Marco Caldin
Tutor:
Josep Pastor Milán
Tesis Doctoral
Departament de Medicina i Cirugia Animals Facultat de Veterinària
Universitat Autònoma de Barcelona 2017
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5 Los doctores JOSEP PASTOR MILÁN, ASTA TVARIJONAVICIUTE y MARCO CALDIN, Profesor Titular del Departament de Medicina i Cirugia Animals de la Facultat de Veterinària de la Universitat Autònoma de Barcelona, Investigadora Posdoctoral de la Facultad de Veterinaria de la Universidad de Murcia y Director Sanitario de la Clinica Veterinaria Privata San Marco en Padua (Italia), respectivamente.
INFORMAN:
Que la memoria titulada AVANCES EN NUEVOS MARCADORES DE DAÑO RENAL EN EL DIAGNÓSTICO Y TRATAMIENTO DE LA LEISHMANIOSIS CANINA, presentada por SAMANTHA GUERRERO CABRERA para la obtención de grado de Doctor en Veterinaria y la Mención de Doctor Internacional por la Universitat Autònoma de Barcelona, ha sido realizada bajo nuestra dirección y, considerándola satisfactoriamente finalizada, autorizamos su presentación para que sea juzgada por la comisión correspondiente.
Y para que conste a los efectos oportunos, firmamos el presente informe en Bellaterra, a 25 de septiembre de 2017.
Firmado
Josep Pastor Milán Asta Tvarijonaviciute
Marco Caldin
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7
A mi madre y a mi padre
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9 Viajar es marcharse de casa, es dejar los amigos es intentar volar volar conociendo otras ramas
recorriendo caminos es intentar cambiar.
Viajar es vestirse de loco es decir “no me importa”
es querer regresar.
Regresar valorando lo poco saboreando una copa, es desear empezar.
Viajar es sentirse poeta, es escribir una carta,
es querer abrazar.
Abrazar al llegar a una puerta añorando la calma
es dejarse besar.
Viajar es volverse mundano es conocer otra gente
es volver a empezar.
Empezar extendiendo la mano, aprendiendo del fuerte,
es sentir soledad.
Viajar es marcharse de casa, es dejar los amigos es intentar volar;
volar conociendo otras ramas recorriendo caminos es intentar cambiar.
Viajar es regresar - Gabriel García Márquez
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11
Agradecimientos
12
13 Esta tesis doctoral es el final de un camino recorrido, que no hubiese sido posible sin la orientación y el apoyo de mis directores a los cuales quiero extender mis agradecimientos:
A Josep Pastor, gracias por aceptarme como alumna, por darme ánimos en los momentos de crisis, por la paciencia en los altibajos y por no perder la esperanza, aunque a veces pareciera un caso perdido. Gracias por todos los conocimientos y enseñanzas compartidas durante mi estancia en el HCV-UAB y en el Laboratorio de Hematología. Hacen parte de los mejores momentos de mi formación profesional.
A Asta Tvarijonaviciute, gracias por su disponibilidad y apoyo durante la elaboración de este manuscrito, y por aportar su asesoría y experiencia para el desarrollo de esta investigación.
A Marco Caldin, gracias por abrirme las puertas de su Clínica, por compartir de primera mano sus conocimientos y una pizca de su mente brillante. Gracias por poner a disposición los medios y la infraestructura sin los cuales esta investigación no habría sido posible.
También quisiera agradecer a las personas que durante estos años han hecho parte directa e indirecta de esta etapa de mi vida:
A José Joaquín Cerón, gracias por su disponibilidad, por aportar su amplia experiencia en investigación para el desarrollo de los estudios y por facilitarme los medios para finalizar esta investigación.
A Tommaso Furlanello, gracias por la participación en sus casos clínicos, por las sesiones de citología y por su infinita disponibilidad y efectividad para resolver problemas.
14 A Paolo Silvestrini, gracias por creer en mí. Su pasión y excelencia en nuestra profesión ha sido el mejor ejemplo y aliciente para trazarme nuevas metas. Su amistad me puso de nuevo con la brújula rumbo España, y ha valido la pena.
Mi gratitud se extiende a todo el equipo del Laboratorio d’Analisi San Marco por su disponibilidad y por permitirme hacer parte de su grupo de trabajo. Agradezco especialmente a Claudia y Chiara por su colaboración con las tareas de laboratorio y a Graziano por contribuir al diseño experimental y al análisis estadístico de la investigación.
A tutti i miei amici de la Clinica San Marco, italiani di nascita o italiani di cuore. Grazie alla vostra diversità ho conosciuto l'Italia da sud a nord.
Mi avete aiutato a innamorarmi dell'Italia. Avete cambiato la mia vita ragazzi, vi porto nel cuore. Tucci, grazie semplicemente della tua amicizia.
Finalmente quiero agradecer a mi familia. Ellos son el pilar de la construcción de mi proyecto de vida.
A mis hermanos Nickolas y Lynna, gracias por su apoyo incondicional. A mi tía Olga, gracias por ser como una madre y brindarme un lugar en su corazón y en su hogar.
A mi madre por su infinita paciencia, compresión y cariño incondicional.
Gracias por ser siempre mi fan número uno, y consentir todos mis proyectos. Por creer en mí, cuando ni yo misma me lo creo. Eres un ejemplo de que todo se puede realizar con un poco de fe en sí mismo.
A mi padre, por su soporte, comprensión y por su continuo aliento. Por ser un ejemplo en la vida académica y profesional, pero sobre todo por ser un padre ejemplar. Este trabajo es para ustedes dos.
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1. Índice
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17
1. Índice 15
2. Resumen 19
3. Abstract 23
4. Abreviaturas 27
5. Introducción 33
6. Objetivos 37
7. Revisión Bibliográfica 41
7.1. Renal disease in canine leishmaniosis 43
7.1.1 Histopathology 44
7.2. Urinary renal biomarkers in canine leishmaniosis 46 7.2.1 Biomarkers of glomerular damage 47 7.2.1.1 Glomerular filtration rate (GFR) 47
7.2.1.2. Serum creatinine (sCr) 49
7.2.1.3. Serum urea nitrogen 52
7.2.1.4. Urine protein:creatinine ratio (UPC) 53 7.2.1.5. Symmetric dimethylarginine (SDMA) 56
7.2.1.6. Immunoglobulins 58
7.2.1.7. Cystatin C (CysC) 61
7.2.2. Biomarkers of tubular damage 63 7.2.2.1.¡-glutamyl transferase (GGT) 63 7.2.2.2. N-acetyl-β-D-glucosamidase (NAG) 64 7.2.2.3. Retinol-binding protein (RBP) 66
7.2.2.4. Clusterin (Clus) 67
18
7.2.3. Biomarkers of inflammation 68
7.2.3.1. C-reactive protein (CRP) 68
7.2.3.2. Ferritin 70
7.2.3.3. Adiponenctin 71
8. Estudios 77
8.1. Estudio I 79
8.2. Estudio II 87
9. Discusión General 107
10. Conclusiones 117
11. Conclusions 121
12. Bibliografía 125
19
2. Resumen
20
21 La leishmaniosis canina (CanL, canine leishmaniosis) es una zoonosis endémica transmitida por vectores. La enfermedad renal es una de las principales complicaciones y causas de mortalidad en la CanL. En la leishmaniosis humana se ha observado disfunción renal tubular asociada con defectos en la concentración de orina y con desequilibrios electrolíticos.
Las pruebas de laboratorio comúnmente utilizadas para evaluar la función renal carecen de una adecuada sensibilidad y especificidad, y además la progresión de la enfermedad renal es frecuentemente asintomática en las etapas iniciales de la CanL. Todo esto hace necesario encontrar nuevos biomarcadores capaces de detectar el daño renal precoz, que puedan ser útiles para diagnosticar y evaluar la progresión y la respuesta al tratamiento de la enfermedad renal secundaria a CanL.
Esta investigación se dividió en dos estudios. En el primer estudio, se realizó la validación analítica de la medición con osmometría de punto de congelación de la osmolalidad urinaria (UOsm, urine osmolality) en una amplia población de perros clínicamente sanos, demostrando un buen desempeño del método. Se establecieron intervalos de referencia en perros jóvenes-adultos y en perros ancianos. También se determinó el efecto de la edad, el sexo y el estado reproductivo sobre la UOsm.
Los resultados mostraron que la concentración de orina disminuye en perros ancianos. No se observó ninguna influencia del sexo, pero se detectó una UOsm menor en las perras castradas en comparación con las intactas, sugiriendo un efecto de la esterilización sobre la concentración de orina.
22 En el segundo estudio, se evaluó la UOsm y las fracciones de excreción de Na, K, Cl y Mg en diferentes estadios de la enfermedad renal en una pequeña población de perros naturalmente infectados con L. infantum.
Los cambios en estos analitos se evaluaron en perros con proteinuria luego de un mes de tratamiento. Todas las fracciones de excreción evaluadas, con excepción de la fracción de excreción de K, probaron ser biomarcadores útiles para detectar disfunción tubular temprana antes de la presentación de azotemia. Sin embargo, sólo la fracción de excreción del Mg mostró una clara evidencia de la mejoría renal después del tratamiento.
Los resultados de esta investigación proveen la base para investigaciones posteriores acerca de la UOsm y las fracciones de excreción of Na, K, Cl y Mg, y su aplicación en el área clínica para el diagnóstico y monitorización del daño renal.
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3. Abstract
24
25 Canine leishmaniosis (CanL) is a vector-borne endemic zoonosis. Renal disease is one of the main complications and a major cause of mortality in CanL. Renal tubular dysfunction has been observed in human leishmaniosis associated with defects in urine concentration and electrolytic disorders.
Most commonly used tests for renal function assessment lack adequate sensitivity and specificity, and as renal disease progression is often asymptomatic in the initial stages of CanL, it is necessary to find new biomarkers able to detect early kidney damage, that could be useful to diagnose, evaluate the progression and the response to therapy of renal disease secondary to CanL.
This research was divided into two studies. In the first study, the analytical validation of freezing point depression measurement of canine urine osmolality (UOsm) was performed in a large population of clinically healthy dogs, showing a good performance. Reference interval were established in young-adult and senior dogs. The effect of age, sex, and reproductive status on UOsm was determined as well. The results demonstrated that urine concentration decrease in older dogs. No influence of sex was observed, but UOsm was lower in neutered than in intact female dogs suggesting an effect of sterilization on urine concentration.
In the second study, the evaluation of UOsm and fractional excretion of Na, K, Cl and Mg at different stages of renal disease was performed in a small population of dogs naturally infected with L. infantum. Changes of these tests after one month of treatment were evaluated in dogs with proteinuria. All evaluated fractional excretions, with exception of fractional excretion of K, proved to be useful markers to detect early tubular dysfunction before the presentation of azotemia. However, only fractional excretion of Mg showed clear evidence for renal improvement after treatment.
26 The results of this research provide a basis for the further investigation about UOsm and fractional excretions of Na, K, Cl and Mg, and their application in clinical settings for renal damage diagnosis and monitoring.
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4. Abreviaturas
28
29 ACVIM American College of Veterinary Internal Medicine
ACE angiotensin-converting enzyme ADH antidiuretic hormone
AKI acute kidney injury ALP alkaline phosphatase ALT alanine aminotransferase APP alanine aminopeptidase
CanL leishmaniosis canina, canine leishmaniosis
CI confidence interval
CKD chronic kidney disease
Cl chloride
Cr creatinine
CV coefficient of variation CVs coefficients of variation
CysC cystatin C
FE fracción de excreción, fractional excretion FEs fracciones de excreción, fractional excretions FECl fractional excretion of chloride
FEIgG fractional excretion of Immunoglobulin G FEIgM fractional excretion of Immunoglobulin M FEK fractional excretion of potassium
FEMg fractional excretion of magnesium FENa fractional excretion of sodium GFR glomerular filtration rate GGT gamma-glutamyltransferase HMW high molecular weight
IgA Immunoglobulin A
IgG Immunoglobulin G
IgM Immunoglobulin M
IL-2 interleukine-2
30 IL-7 interleukine-7
IL-18 interleukine-18
IRIS International Renal Interest Society;
Sociedad Internacional de Interés Renal
K potassium
kDa Kilodaltons
KIM-1 kidney injury molecule-1 LDH lactate dehydrogenase LMW low molecular weight LOD limit of detection LOQ limit of quantification
Mg magnesium
MHC major histocompatibility complex MMW middle-molecular weigh
mOsm/kg milliosmoles per kilogram NAG N-acetyl-b-D-glucosaminidase
NGAL neutrophil gelatinase-associated lipocalin r2 adjusted correlation coefficient
RBP retinol-binding protein RIs reference intervals sCysC serum cystatine C sCr serum creatinine SD standard deviation
SDMA symmetric dimethylarginine Se serum electrolyte concentration SG specific gravity
sNGAL serum neutrophil gelatinase-associated lipocalin THP Tamm–Horsfall protein
TTR transthyretin
TXB2 thromboxane B2
uGGT urinary gamma-glutamyltransferase
31 uClus urinary clusterin
uCysC urinary cystain C
uCPR urinary C-reactive protein uRBP urinary retinol-binding protein uCr urinary creatinine
Ue urinary electrolyte concentration uFer urinary ferritin
uIgA urinary Immunoglobulin A uIgG urinary Immunoglobulin G uIgM urinary Immunoglobulin M
uNAG urinary N-acetyl-b-D-glucosaminidase
uNGAL urinary NGAL
UOsm urine osmolality
UPC urine protein:creatinine ratio USG urine specific gravity
XLHN X-linked hereditary nephropathy
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33
5. Introducción
34
35 La leishmaniosis canina producida por Leishmania infantum (sin.
Leishmania chagasi en Latinoamérica) es una de las enfermedades protozoaria, zoonótica y trasmitida por vectores, más importantes a nivel mundial por su carácter potencialmente fatal (Solano-Gallego et al. 2011; Paltrinieri et al. 2016). La leishmaniosis canina es endémica en Europa, Asia, Norte de África y Sur América, y es una enfermedad emergente en Norte América (Duprey et al. 2006, Maia et al. 2010). En la cuenca mediterránea, se estima que entre el 65-80% de los perros han tenido contacto con Leishmania (Solano-Gallego et al. 2011).
La enfermedad renal, puede ser una de las únicas manifestaciones de la leishmaniosis canina. Se caracteriza por un curso lento, progresivo y frecuentemente asintomático, siendo una de las principales causas de mortalidad de los perros con Leishmania. (Solano-Gallego, et al. 2011).
Con menor frecuencia puede presentarse de forma aguda e hiperaguda.
La disfunción renal en la leishmaniosis canina, es provocada principalmente por la respuesta inflamatoria persistente desencadenada por el depósito de inmunocomplejos a nivel glomerular y tubular causando glomerulonefritis, glomeruloesclerosis y nefritis intersticial (Paltrinieri et al. 2016). Las lesiones renales afectan la filtración glomerular y la funcionalidad tubular e intersticial, alterando la capacidad de concentrar la orina, y la reabsorción y la excreción renal de solutos como las proteínas y los electrolitos (Hokamp and Nabity, 2016).
Tanto en humanos como en perros, los métodos diagnósticos convencionales, como la creatinina y la urea séricas, y la densidad urinaria determinada por refractometría, carecen de sensibilidad y/o especificidad para identificar la enfermedad renal cuando el daño es incipiente, y poseen una menor capacidad para determinar el origen glomerular o tubular de dicho daño.
36 El diagnóstico precoz de la enfermedad renal en la leishmaniosis canina, podría permitir la instauración de medidas de prevención y de un tratamiento adecuado, para evitar la progresión y empeoramiento de las lesiones renales, así como para aumentar las probabilidades de supervivencia de los animales afectados.
Por dichas razones, se hace necesaria la búsqueda de nuevos marcadores biológicos, capaces de detectar un mínimo de daño renal.
Recientemente se ha incluido la determinación de la dimetil arginina simétrica sérica y/o plasmática en la clasificación de la Sociedad Internacional de Interés Renal (IRIS, International Renal Interest Association) de la enfermedad renal crónica, demostrando la importancia y potencial de la investigación en los nuevos biomarcadores de daño renal. La detección de marcadores biológicos más sensibles y específicos de lesión a nivel glomerular o tubular, pueden convertirse en una herramienta útil para detectar estadios iniciales de daño renal, valorar la severidad de las lesiones y la recuperación de la funcionalidad, la evolución y la respuesta, al tratamiento en la enfermedad renal secundaria a la leishmaniosis canina.
En esta investigación se validó la medición de la osmolalidad urinaria por medio de la osmometría de punto de congelación, se establecieron intervalos de referencia según la edad, y se determinó el efecto de la edad, el género y el estado reproductivo, sobre la concentración urinaria en perros sanos. Posteriormente, se estudiaron los cambios en la osmolalidad urinaria y las fracciones de excreción de sodio, potasio, cloro y magnesio en diferentes estadios de enfermedad renal en perros naturalmente infectados con Leishmania, y la dinámica de dichos analitos en el seguimiento de la evolución de la enfermedad y la respuesta al tratamiento.
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6. Objetivos
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39 1. Realizar la validación analítica de la osmometría de punto de congelación como método para medir la osmolalidad urinaria en perros sanos.
2. Establecer intervalos de referencia de la osmolalidad urinaria según la edad en perros jóvenes-adultos, y en perros ancianos.
3. Evaluar los efectos de la edad, sexo, y el estado reproductivo en los valores de la osmolalidad urinaria en perros sanos.
4. Determinar los cambios de la osmolalidad urinaria, y las fracciones de excreción de sodio, potasio, cloro y magnesio, en diferentes estadios de la enfermedad renal secundaria a la leishmaniosis canina.
5. Investigar los cambios en los valores de la osmolalidad urinaria y las fracciones de excreción de sodio, potasio, cloro y magnesio, al momento del diagnóstico y después de un mes de tratamiento en diferentes estadios de la enfermedad renal secundaria a la leishmaniosis canina.
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41
7. Revisión
Bibliográfica
42
43 7.1 Renal disease in canine leishmaniosis
Nephropathy in CanL is a multifactorial disorder that undergoes chronic evolution. It has been proposed that renal disease in CanL is mainly produced due to high serum concentration of immune complexes and its tissue deposition, vasculitis secondary to activation of the complement system and antihistone antibodies presented in circulation within the glomeruli causing glomerulonephritis (Torrent et al. 2005; Koutinas and Koutinas, 2014).
However, one study by Costa et al. (2010) proposed that immunoglobulins and complement play no role in the pathogenesis of advance stages of glomerulonephritis. As an alternative, these authors suggested that perpetuation and progression of the underlying glomerular disease in CanL was associated to hypercellularity secondary to migration of inflammatory cells and/or decreased apoptosis. The presence of Leishmania antigen in mesangial cells that probably guides inflammatory infiltrate of CDT+4 cells, and the decrease in apoptosis mediated by low production of tumoral necrosis factor, both contributes to hypercelullarity. Adhesion molecules as P-selectin and intercellular adhesion molecule-1 that migrate with inflammatory cells help to determine the proliferative pattern of glomerulonephritis (Costa et al.
2010).
Recently, severity of the glomerular lesion in CanL has been related to an increase in the inflammasone complex characterized by the increase in glomerular nucleotide-binding domain leucine-rich repeat-containing- like receptor family, pyrin domain containing 3 and autophagosome- associated microtubule-associated protein 1 light chain 3 (Esch et al.
2015). Inflammasome activity helps in host defense by activation of a rapid inflammatory response and restriction of pathogen replication, but also contributes to the development and progression of chronic
44 pathologies such as renal disease due to Leishmania infection (de Zoete et al. 2014).
Renal disease in CanL can be presented in the forms of acute kidney injury (AKI) to chronic kidney disease (CKD) and be asymptomatic or symptomatic. The asymptomatic presentation often course with proteinuria. Progressive proteinuria could lead to tubular excretory dysfunction, reduction or increment of glomerular filtration rate (GFR) and systemic hypertension. Intense proteinuria could cause muscular wasting and cachexia, and occasionally pulmonary thromboembolism.
Severe proteinuria and end stage kidney disease are the principal cause of death in chronic Leishmania cases in dogs (Koutinas and Koutinas, 2014).
Renal tubular dysfunction in Leishmania infection has been observed in asymptomatic humans. Its seems to be related to urinary concentration and/or acidification deficits, presented before a decrease GFR and an increase in serum creatinine (sCr) (Silva Junior et al. 2014). Urinary concentration deficit has been associated with lower expression of aquaporine 2 and an increase in the expression of cotransporter Na-K- 2Cl as compensatory mechanism in renal tubules producing lower urine osmolality (UOsm). Acidification deficit has been related to abnormalities in the renal transporters implied in acid-base regulation including an increased expression of Na/H exchanger 3 in the proximal tubule, and H-ATPase and pendrin in the distal tubule. Renal tubular dysfunction has also been related to serum and urinary electrolytic disturbances in humans (Lima Verde et al. 2007; Oliveira et al. 2011;
Silva Junior et al. 2014).
7.1.1. Histopathology
Histopathological studies about CanL demonstrate glomerular lesions in all animals involved (Costa et al. 2003; Zatelli et al. 2003). Chronic
45 glomerulonephritis represents the outcome of several types of glomerulonephritis probably because of the long evolution of the disease in dogs, even when the parasite was probably eliminated from the tissue (Costa et al. 2003).
Glomerular lesions in CanL have been histologically classified as minor glomerular abnormalities, focal segmental glomeruloesclerosis, proliferative glomerulonephritis, membranous glomerulonephritis, membranoproliferative glomerulonephritis, crescentic glomerulonephritis and chronic glomerulonephritis (Costa et al. 2003;
Zatelli et al. 2003).
Glomerulonephritis and diffuse mesangial proliferative glomerulonephritis are the most frequent patterns observed in CanL in Brazil and Italy (Costa et al. 2003; Zatelli et al. 2003). In Spain (Nieto et al. 1992) and France (Benderitter et al. 1988), diffuse membranoproliferative glomerulonephritis was the most frequent histopathologic pattern observed in naturally infected dogs (Costa et al, 2003). The variation presented between countries could be related to the number of animals in the studies and different species of Leishmania involved in each country (Hosein et al. 2017).
Focal segmental glomerulosclerosis and ultrastructural glomerular changes in normal histology samples have been found in leishmaniotic dogs, non proteinuric to borderline proteinuric, with normal plasma Cr (Costa et al. 2003).
Costa et al. (2003) observed tubular changes in 96% (53 of 55) and interstitial nephritis in 78% (43 of 55) of the animals studied. Zatelli et al. (2003) observed tubulointerstitial histologic changes less frequently.
Inflammation affected principally the renal cortex and was characterized by organized foci with lymphocytes, plasma cells, and scarce histiocytes and polymorphonuclear neutrophils. Diffuse inflammatory infiltration
46 was severe, less frequent and was characterized by small clusters to massive foci and intertubular cords spreading in areas of fibrosis. Renal cortex was the most affected by interstitial nephritis. In the Costa et al.
study, even in the cases when there was inflammatory infiltrate in the medulla (26%), also the lesions were more severe in the cortex. The most frequent tubular change was vacuolar and hyaline degeneration, also presented without interstitial inflammatory infiltration and atrophy.
It has been observed that isolated tubules with severe inflammatory foci develop necrosis. These changes are potential causes of renal dysfunction in CanL (Costa et al. 2003).
It has been proposed that tubulointersticial histopathologic changes are secondary to glomerular pathology, immune complex mediated inflammation and renal interstitium fibrosis (Koutinas and Koutinas, 2014), rather than a primary lesion due to Leishmania infection.
7.2 Urinary renal biomarkers in CanL
Biological markers known as biomarkers, are valuable tools to identify normal or pathogenic processes, or the response to treatment, and are helpful to determine patient status. Biomarkers are often used to diagnose and measure a pathological condition and prognosis of the disease. They are not necessarily involved or play a role causing the disease process (Tesch et al. 2010).
To be useful in the clinical setting, an ideal renal biomarker should be measured easily, non-invasively, accurately and reproducibly. Renal biomarkers could localize kidney injury (i.e., glomerular level, tubular level or both), sensitively indicate renal injury and predict its severity, differentiate renal injury from pre-and post-renal causes, identify or differentiate specific types of renal injury or kidney disease, diagnose
47 non renal injury, and monitor the kidney response to treatment. Renal biomarkers should provide useful and cost-effective clinical information that is easy to interpret, gives additional information to conventional clinical parameters, and it is applicable across a variety of populations (Tesch et al. 2010; De Loor et al. 2013).
Urine is one of the most promising biological fluids to research for earliest biomarkers of kidney injury because is easy to obtain and is closely related to the kidney. Renal biomarkers in urine could be determined by the evaluation of specific proteins or proteome profile analysis that contributes with a global overview of proteins and peptides in urine (De Loor et al. 2013). Changes in renal handling of water and other particles (urine osmololality) and variation of urine excretion of solutes such as electrolytes (fractional excretions) are also useful as renal biomarkers.
7.2.1. Markers of glomerular damage or dysfunction
7.2.1.1 Glomerular filtration rate (GFR)
Glomerular filtration rate is defined as the volume of ultrafiltrate produce per unit of time by glomerular filtration. GFR is considered one of the best methods to evaluate kidney function (Brown and Lefebvre, 2008). GFR could be affected by several extra-renal factors including sex, age, breed, weight, dietary protein intake, hydration status, sodium balance, exercise, and day-to-day circadian rhythm. The most commonly used GFR standardization method is the body weight in kg and the body corporal surface (Brown and Lefebvre, 2008; Von Hendy- Willson and Pressler, 2011).
48 Direct determination of GFR has been performed using the clearance of molecules freely filtered by the glomerulus with minor or no secretion and tubular reabsorption, and is not bound to plasma proteins (Brown and Lefebvre, 2008). Indirect estimation of GFR is commonly done in the clinical setting by the measurement and interpretation of serum biomarkers as Cr (Von Hendy-Willson and Pressler, 2011).
The standard method to measure GFR is renal clearance of inulin. The filtered load is equal to the total amount of this marker that undergo urinary elimination over a period. Nonetheless, this method is impractical and time-consuming because to perform the measurement is necessary the maintenance of a constant plasma concentration by constant infusion of the marker, the accurate determination of total urine volume using metabolic cages or indwelling catheters, and precision and accuracy to measure the marker (Brown and Lefebvre, 2008). Plasma clearance methods have also been used to measure GFR by determining the reduction in plasma concentration of a marker over time. The plasma concentration-versus-time curve is determined by obtaining multiple plasma samples at set time intervals for a predetermined length of time (Brown and Lefebvre, 2008). Clearance of molecules such as Cr, iohexol and radiolabeled markers have also been used to determine GFR in dogs. Other techniques include the application of contrast-enhanced computed tomography to indirectly measure an injected marker by determining it renal uptake (Von Hendy-Willson and Pressler, 2011). However, lack of standardization of protocols and complications in the reproducibility of results make the GFR measurement in dogs difficult to apply in the clinical setting (Von Hendy-Willson and Pressler, 2011).
GFR is commonly decreased by the reduction of renal artery pressure secondary to hypotension or effective circulating volume depletion (Von Hendy-Willson and Pressler, 2011). Renal mass loss of 5/6 has been
49 related to a 65% decrease in GFR, experimentally in dogs. AKI is generally associated with an acute decrease of GFR, and CKD with it progressive decline (Brown and Lefebvre, 2008).
One study evaluated the GFR changes in dogs naturally infected with Leishmania, showing that most of the proteinuric non azotemic or mildly azotemic dogs had low GFR, but glomerular hyperfiltration occurs in few dogs. Glomerular hyperfiltration was probably due to early kidney injury and/or the result of systemic hypertension. In consequence, GFR measurement could be helpful to detect early renal damage in CanL (Cortadellas et al. 2008).
7.2.1.2. Serum Creatinine (sCr)
Creatinine is a heterocyclic, nitrogenous compound with a low molecular weight (113 kDa) produced as the result of normal muscle metabolism (Relford et al. 2016). It is originated by spontaneous, irreversible, nonenzymatic, internal dehydration of creatine and dephosphorylation of phosphocreatine. Creatine and Cr are originated from endogen glycine, arginine, and methionine. A relatively minor source of Cr is ingested during consumption of animal tissue and absorbed from the intestines. Daily and almost constant production of Cr occurs in the kidney, and accounts for 2% of the total body pool of creatine (Braun et al. 2003). In dogs, endogenous Cr production accounts for about 90%
of sCr (Concordet et al. 2008).
Under normal conditions, plasma Cr is filtered freely through the glomerulus and is not reabsorbed in the tubules. Consequently, its serum concentration is the same to the glomerular filtrate. However, in the proximal renal tubules Cr is weakly secreted, being most prominent in males but without significance even in male dogs with CKD (Braun et
50 al. 2003).
Urinary elimination of Cr is constant over time. Urinary creatinine (uCr) values do not have daily variations, but could increase or remain stable after meals. Inter-individual variation of 24 hours uCr has been observed probably due the diverse protein percentage in dietary intake, accuracy of the measurements and changes in urine dilution/concentration (Braun et al. 2003).
Serum creatinine is influenced by age being significantly lower in puppies than in adult dogs (Rørtveit et al. 2015), and is higher in large breeds (Braun and Lefebvre, 2008). Weight and/or muscle mass is the most probable reason in both cases (Concordet et al. 2008). No effect of gender has been reported in creatinine urinary elimination (Hokamp and Nabity, 2016) but moderately higher plasmatic concentrations has been observed in male dogs (Braun and Lefebvre, 2008). Also, sCr concentration could by influenced by hydration state, physical effort, secondary diminution of GFR by nephrotoxic drugs, alter renal hemodynamics by drugs and extracellular dehydration (Braun et al 2003).
Serum creatinine (sCr) values increases above the reference interval with at least 75% of nephron mass loss. Studies in experimental partially nephrectomized dogs demonstrate that this percentage corresponds to 35-60% of decrease in renal function. The difference between mass loss and renal function is in part due to compensatory mechanisms as renal hypertrophy (Hokamp and Nabity, 2016).
In practice, sCr concentration is commonly used as an indirect marker of GFR in dogs because it increases exponentially as GFR declines (Relford et al. 2016). Increment in sCr has been demonstrated in CKD and AKI, but its concentration is not useful to differentiate between them. However, sCr is one of the principal markers used by the IRIS
51 guidelines to classify AKI and CKD, and determine the presence and the severity of the azotemia in dogs (Table 1). Even sCr concentration increases according to the progression of CKD, it has a marked inter- individual variation, being a poor predictor of changes in GFR in some dogs (Brown et al. 2003). Has been reported that sCr could be more sensitive for detecting decreased renal function than serum urea nitrogen (Hokamp and Nabity, 2016) but is less sensitive than other renal markers to detect early renal damage (Paltrinieri et al. 2016). It seems that sCr could be a better biomarker for monitoring changes when the renal disease is established or to assess the efficiency of the treatment (Braun et al. 2003; Ghys et al. 2014).
Table 1. Staging of CKD based on blood creatinine concentration*.
Stage Blood creatinine Comments
At risk <1.4 At risk
1 <1.4 Non azotemic. Other renal abnormalities such as
inadequate urinary concentration without non renal cause or proteinuria of renal origin
2 1.4-2.0 Mild renal azotemia. Clinical signs mild or absent 3 2.1-5.0 Moderate renal azotemia. Many extrarenal clinical signs
may be presented
4 <5.0 Increasing risk of systemic clinical signs and uremic crises
*Adapted from Elliot J, Watson AD. Chronic kidney disease: International Renal Interest Society staging and management. In: Bonagura JD, ed. Twedt DC. Kirk’s current veterinary therapy.
15th ed. Chapter 189. United States of America, Missouri: Saunders, 2014:857-863.
Moreover, sCr evaluation is used to classify the severity of CanL by the LeishVet guidelines, according to the IRIS staging of CKD mentioned above (Solano-Gallego et al. 2011).
In renal disease due to Leishmania infection, significant negative
52 correlation between sCr and GFR has been observed in non azotemic and proteinuric dogs. However, some of these dogs had high GFR indicating glomerular hyperfiltration, that could be observed in early glomerular damage and predicts nephropathy development. This discrepancy shows that sCr could mislead changes in GFR and its lack of sensibility to detect early renal damage in non azotemic, proteinuric dogs (Cortadellas et al. 2008). Also, has been reported that sCr values showed no changes associated with improvement in proteinuria after one month of treatment, in non azotemic proteinuric dogs with renal disease due to leishmaniosis (Pardo-Marín et al. 2017).
7.2.1.3. Serum urea nitrogen
Urea is a nitrogenus end-product, with a molecular mass of 60.06 g/mol, water-soluble and neutral charged molecule. Biosynthesis of urea occurs mainly in the liver from ammonia generated by catabolism of dietary proteins in the intestines, or from endogenous tissue proteins. Ammonia originated by the colon flora from protein that escapes absorption in the small bowel and by recycled urea also contribute to the urea cycle passing into the liver through the portal circulation (Wang et al. 2014).
Urea biosynthesis could be regulated by hormones such as insulin and glucagon, growth hormone and insulin growth factor-1, and could be affected by endogens and exogenous glucocorticoids. Blood urea values are also affected by tissue breakdown, high protein intake, and major gastrointestinal hemorrhage (Wang et al. 2014).
Serum/plasma urea nitrogen values are decreased until 50% in dogs between birth and 1-2 months. No gender effect has been reported.
Lipemia, hemolysis and ictericia interfere with urea nitrogen measurements (Braun and Lefebvre, 2008).
53 About 90% of urea is eliminated in urine mostly by glomerular filtration and in lower proportion by tubular secretion in the thin segment of Henle (Hosten, 1990; Wang et al. 2014). Almost half of the urea is reabsorbed in the proximal tubules. The amount of urea resorbed in the collecting ducts is dependent on the permeability and the tubular concentration of urea in this segment. Both factors are affected by the presence of antidiuretic hormone (ADH). The reabsorption of urea in the medullary collecting ducts and its intrarrenal recycling contribute to create the osmotic gradient, critical concentrating urine (Hosten, 1990).
A small amount of urea is lost through the gastrointestinal tract, lungs and sweat (Wang et al. 2014).
In the normal kidney, at high flow rates, approximately 40% of filtered urea is reabsorbed. At low flow rates, approximately 60% of filtered urea is reabsorbed and added back to the blood urea concentration.
This explains the high urea nitrogen levels seen when GFR decrease by any cause. Increment in urea nitrogen could be secondary to pre-renal, renal and post-renal causes, and due to increased catabolism and/or protein digestion. Decreased urea nitrogen values could be the result of decreased protein intake or protein anabolism, increased excretion as in any case of polyuria and decrease in production as in liver failure (Braun and Lefebvre, 2008).
Serum/plasma urea nitrogen is considered a screening test of renal function when is interpreted together to sCr values. However, urea measurement is less specific than sCr for the diagnosis and management of CKD (Braun and Lefebvre, 2008) and it is influenced by dehydration, gastrointestinal hemorrhage or increase in protein catabolism.
54 7.2.1.4. Urine protein: creatinine ratio (UPC)
The glomerular filtration barrier is the main mechanism for preventing proteinuria. This barrier allows free filtration of proteins <40 kDa (LMW, low molecular weight) to filter freely. Middle molecular weight (MMW) proteins are largely restricted, and high molecular weight (HMW) proteins (>100 kDa) are almost completely restricted from glomerular filtration. As well, the proximal tubular epithelial cells are responsible to reabsorb proteins normally present in the urinary filtrate, to prevent proteinuria (D’Amico and Bazzi, 2003).
Proteinuria is consequence of the impairment in the glomerular barrier permeability that allows filtration of albumin and HMW proteins.
Abnormally filtered proteins increased the load allowed by the renal tubules and saturate the reabsorption mechanism. This situation induces toxic damage in the proximal tubules leading to an insufficient protein reabsorption, mainly of the LMW ¡proteins (D’Amico and Bazzi, 2003). The detection of increase in protein excretion is useful to the diagnosis and prognosis in the early detection and confirmation of renal disease. Its quantification could be useful to assess the effectiveness of therapy and the progression of the disease (Price et al. 2005).
The urine protein:creatine ratio (UPC) is a measurement that allows to quantify proteinuria (Paltrinieri et al. 2016) in spot samples, based on the assumption that excretion of Cr and protein is almost constant throughout the day when the GFR is stable (Price et al. 2005). The value of UPC could be affected by contamination from the low urinary tract during collection, and by the presence of active urinary sediment (Paltrinieri et al. 2016). In dogs, long-term glucocorticoid therapy produced a regular increase of UPC from 0.5 at 2 weeks, to above 1 after 4 weeks (Braun and Lefebvre, 2008).
55 IRIS guidelines (Table 2) and the American College of Veterinary Internal Medicine (ACVIM) proteinuria consensus statement, recommend cut-off points or decision limits to evaluate proteinuria in dogs with renal disease (Littman et al. 2013).
Table 2. Substaging of CKD based on urine protein:creatinine values*
UPC values Substaging
>0.2 Non proteinuric 0.2-0.5 Borderline proteinuric
>0.5 Proteinuric
*From Elliot J, Watson AD. Chronic kidney disease: International Renal Interest Society staging and management. In: Bonagura JD, ed. Twedt DC. Kirk’s current veterinary therapy. 15th ed.
Chapter 189. United States of America, Missouri: Saunders, 2014:857-863.
UPC provides an indication of altered glomerular permselectivity, as has been shown in X-linked hereditary nephropathy (XLHN), a model of glomerular disease in dogs (Nabity et al. 2012). UPC values ≥2 could be indicative of glomerular proteinuria, while values <2 are often present in tubular proteinuria. Nonetheless, it has been observed that some proteinuric dogs with UPC <2 had primary glomerular damage confirmed by renal biopsy. Consequently, UPC values could not always differentiate glomerular from tubular damage (Cianciolo et al. 2016).
Recently, has been reported method-dependent analytic variability (pyrogallol red molybdate versus Coomassie brilliant blue) in the measurement of total proteinuria that could affect the final interpretation of the UPC ratio and reduce the ability to correctly classify dogs with UPC < 0.2 (Rossi et al. 2016).
Otherwise, persistent renal proteinuria quantify by UPC levels, could be an early indicator and a negative prognostic factor of CKD in dogs
56 (Hokamp et al. 2016). High UPC values have been considered a risk factor for the progression of nephropathy (Paltrinieri et al. 2016) and a risk factor of mortality in AKI (Brown et al. 2015).
Proteinuria should be assessed in any dog suspected or diagnosed with Leishmania infection, according to the ACVIM consensus for glomerular diseases (Littman et al. 2013). Usually, urine dipstick is a good screening test to first assess proteinuria, but UPC is preferred to diagnose and follow-up CanL. UPC values are used to classify the severity of the disease by LeishVet guidelines (Solano-Gallego et al.
2011), according to proteinuria classification by IRIS guidelines.
Proteinuria also has been proposed as a negative prognostic factor in dogs with leishmaniosis (Paltrinieri et al. 2016)
One study in CanL at different stages of renal disease reported a significant correlation between UPC and measured GFR values.
However, it seems that only a small proportion of the variability in GFR can be predicted from changes in UPC (Cortadellas et a. 2008).
Frequently, UPC values are used to monitor the treatment in CanL. One study demonstrates the reduction in the magnitude of proteinuria at diagnosis, after one month of treatment in sick dogs [stage C]
(Pierantozzi et al. 2013).
7.2.1.5. Symmetric dimethylarginine (SDMA)
Symmetric dimethylarginine (SDMA) is a low molecular methylarginine (202 g/mol), positive charged and produced by obligate post- translational modification and methylation of arginine residues of various proteins in the nucleus of all cells. Free methylarginines are released into the cytosol after proteolysis and pass to the bloodstream.
57 SDMA has an integral role in basic cellular metabolism and is mainly excreted by glomerular filtration (90%) (Relford et al. 2016; Hall et al.
2016).
In dogs, SDMA is not affected by breed, gender or by muscular mass like sCr (Relford et al. 2016; Dahem et al. 2017). SDMA increases by age as GFG decreases, when renal function declines. Hemoglobin, lipids, bilirubin, arginine, monomethylarginine, asymmetric dimethylarginine, and homocitrulline did not interfere with SDMA measurement in humans and dogs (Relford et al. 2016).
SDMA values did not change with acute inflammatory response, hepatic disease, cardiovascular disease or diabetes without concurrent renal disease in humans (Relford et al. 2016). In dogs, there was no correlation between liver enzymes (ALT, ALP, GGT) and cardiac biomarkers (N-terminal pro–brain natriuretic peptide) (Relford et al.
2016).
SDMA has been proposed as a renal biomarker of glomerular damage because its concentration is highly and inversely correlated with GFR (Dahlem et al. 2017). In XLHN has been demonstrated that, SDMA consistently detected <30% loss of renal function using either a general reference interval or serial measurements, being useful to diagnose and monitor the renal disease (Nabity et al. 2015).
SDMA accurately and precisely estimated GFR, being more sensitive than sCR in humans and dogs (Relford et al. 2016). Higher SDMA concentrations has been observed in dogs with renal azotemia compared to healthy dogs. However, no difference between SDMA values in AKI and CKD dogs was shown. The correlation between SDMA and sCr values in AKI is lower than in dogs with CKD, probably because the greater effect of muscular mass loss on sCr in CKD. Also, SDMA/Cr was higher in dogs with CKD compared to AKI ones. A SDMA/Cr ratio
58
>10 has been reported to give a poor prognosis in CKD (Dahlem et al.
2017).
SDMA is a more sensitive marker of reduced renal function than sCr in CKD (Fleck et al. 2003) being useful to detect early stages of CKD without increase in sCr in humans and dogs (Relford et al. 2016).
Trending of sCr and SDMA could be a useful tool in the absence of baseline values, to identify early changes in renal function. However, it seems that proteinuria appears first than changes in SDMA concentrations in the presence of renal dysfunction (Nabity et al. 2015).
Recently, serum SDMA has been evaluated in CanL with renal disease.
It has been reported that SDMA levels did not change when renal proteinuria improves after 1 moth of treatment. In these cases, UPC instead of SDMA would be the recommended test to monitor the therapy (Pardo-Marín et al. 2017).
7.2.1.6. Immunoglobulins
Immunoglobulins are involved in host defense mediated by antibodies.
They are high weight glycoproteins produced by plasma cells in bone marrow, lymph nodes and spleen. The monomeric form of immunoglobulin M (IgM), G (IgG) and A (IgA) weight 900 kDa, 150 kDa and 160 kDa each one. IgA has a dimeric and a polymeric form (Hokamp and Nabity, 2016).
These immunoglobulins cannot pass through the normal glomerular filtration barrier because of their weight. When glomerular injury occurs, they initiate to appear into the urinary filtrate (Hokamp and Nabity, 2016). Urinary IgG (uIgG) concentration has been reported unaltered by hematuria, hemoglobinuria, pyuria and/or urinary tract infection in dogs (Hokamp and Nabity, 2016).
59 Increase in serum IgG and IgA on Western blot and uIgG:creatine ratio (uIgG/Cr) has been observed in AKI secondary to babesiosis (Babesia rossi) and leptospirosis. High levels of uIgG/Cr correlated positively to proteinuria, have been reported in dogs with pyometra associated to glomerular damage. Instead, low values of urinary IgA (uIgA) has been observed in those dogs. There have also been high levels of uIgG/Cr suggesting glomerular dysfunction in dogs with hyperadrenocorticism and snake envenomation (Hokamp and Nabity, 2016).
High concentration of uIgG and uIgM have been shown in dogs with CKD (Hokamp et al. 2016). uIgG/Cr progressively increased in CKD secondary to XLHN, compared to healthy dogs of the same age. Also, uIgG/Cr had tendency to increase before UPC in this disease, contrary to other nephropathies where uIgG/Cr appearance is observed at the same time of proteinuria. IgG has a strong correlation with UPC values showing its ability to indicate a dysfunction in glomerular permeability (Nabity et al. 2012). uIgG/Cr has been also positive correlated to histopathological findings of glomerular and tubulointerstitial damage in dogs with XLHN (Nabity et al. 2012).
Likewise, increments in uIgG/Cr and uIgM/Cr has been positively correlated to immune complex-mediated glomerulonephritis (Nabity et al. 2012, Hokampt et al. 2016). Also, increases in uIgM/Cr concentrations provided the best indication of ultrastructural glomerular damage compared to other biomarkers of renal disease, and it was related to increase risk of renal failure and death in both humans and dogs (Hokamp et al. 2016). Lower uIgM/c has been observed in juvenile
nephropathies, non-immune complex-mediated
glomerulonephropathies, and primary tubular disease (Hokamp et al.
2016).
In addition, fractional excretion of IgM (FEIgM) and IgG (FEIgG) has
60 been evaluated in proteinuric CKD dogs, showing that fractional excretion of immunoglobulins was better than its urine concentration to assess tubular damage. FEIgG has shown a strong correlation with histological tubular dysfunction, while FEIgM was equally correlated to histological glomerular and tubular damage (Hokamp et al. 2016).
On the other hand, uIgG (Solano-Gallego et al. 2003) and uIgA (Solano-Gallego et al. 2003; Zaragoza et al. 2003; Todolí et al. 2009) have been found in proteinuric dogs with Leishmania infection. The presence of urine antibodies in CanL is mainly due to free filtration of immunoglobulins secondary to glomerular damage (Solano-Gallego et al. 2003). In the case of IgA, has been proposed that a small proportion corresponds to local production associated with tubulointerstitial nephritis or lesions in other urinary or genital organs (bladder, urethra and/or prostate) (Todolí et al. 2009).
Lower concentrations of uIgA than uIgG have been found in CanL, even in the presence of severe glomerular damage, probably because of lower IgA than IgG serum levels (Zaragoza et al. 2003). There has been reported a lower correlation of uIgA to proteinuria, sCr and serum urea in comparison to uIgG correlation, that shows less sensitivity of uIgA to evaluate renal damage in CanL (Todolí et al. 2009).
Recently, it has been reported that uIgG/Cr increase according to the development of renal disease (Pardo-Marín et al. 2017), and that is positive correlated to proteinuria progression in CanL (Solano-Gallego et al. 2003; Pardo-Marín et al. 2017). Also, uIgG/Cr decrease has been associated to improvement of proteinuria after one month of treatment (Pardo-Marín et al. 2017) in cases without changes in baseline sCr and serum SDMA. The magnitude of its decrease was higher than other biomarkers of glomerular damage such as uCPR/Cr (Pardo-Marín et al.
2017).
61 7.2.1.7. Cystatin C (CysC)
Cystatin C (CysC) is a non-glycosylated protein from the superfamily of cysteine protease inhibitors, positive charged and with a LMW (13kDa).
It is produced by all nucleated cells at a constant rate and released during phagocytosis acting as a proteinase inhibitor to mediate inflammation (García-Martínez et. al 2015; Hokamp and Nabity, 2016).
CysC is freely filtered in glomeruli and completely absorbed and catabolized by the proximal tubular cells without tubular reabsorption or secretion (Ostermann and Joannidis, 2016). However, it has been reported that CysC can be found in small quantities in the urine with normal renal function in humans (Ghys et al. 2014). An increase in CysC has been specifically related with proximal renal tubular damage (Hokamp and Nabity, 2016).
Some extra-renal factors have been suspected to alter the production rate of serum CysC (sCysC), such as systemic inflammation, thyroid dysfunction, glucocorticoid disorders, corticosteroid therapy and HIV disease in humans (Ghys et al. 2014; Ostermann and Joannidis, 2016).
As well, it values could be affected by hyperbilirubinemia and hypertriglyceridemia (Ostermann and Joannidis, 2016). However, significantly higher CysC has been observed in dogs with CKD compared to dogs with non renal diseases like immune-mediated, endocrine, dermatologic, cardiologic and neoplastic disorders (Ghys et al. 2014).
High levels of CysC were related with malignancies like melanoma and colorectal neoplasia in humans. Instead, seems CysC is not influenced by inflammation or neoplasia in dogs (Wehner et al. 2008; García- Martínez et al. 2015).
Serum CysC values could identify the presence of AKI one or two days
62 earlier than sCr concentration in humans with ≥2 predisposing factors of AKI. In dogs, the rol of sCysC in AKI is controversial, and it seems it could not be a sensitive indicator of decrease GFR, but higher values have been observed in severely ill dogs with hypovolemic-shock compared to healthy dogs probably related to decrease GFR (Ghys et al.
2014).
In humans and dogs, CysC is better than sCr to detect renal dysfunction related to decreased GFR in CKD. Also, in human CKD allows a better mathematical estimation of GFR (Ghys et al.2014). In dogs, it has been reported that sCysC has better sensitivity and higher negative predictive value compared to sCr to detect a diminution in GFR related to early kidney injury (Wehner et al. 2008).
Urine cystatin C (uCysC) values are much lower in healthy humans than in individuals with renal tubular damage. Also, higher uCysC levels could be useful to differentiate cases of proteinuria secondary to proximal tubular damage from cases of proteinuria without renal tubular injury in humans (Ghys et al. 2014). However, massive proteinuria could inhibit the tubular reabsorption of CysC, leading to increase in uCysC that could underestimate tubular function. In consequence, it is important to evaluate total proteinuria when uCysC is measured (Ghys et al. 2014). Also, uCysC values could be useful to predict renal replacement requirements in patients with non-oliguric ATN, but could not differentiate between AKI and CKD in humans (Ghys et al. 2014). In dogs, uCysC and urine cystatin C:creatinine ratio (uCysC/Cr) are useful to differentiate between animals without renal disease and dogs with renal disease and azotemia (Monti et al. 2012).
Urine cystatin C values tend to increase according to severity of CKD in dogs with Leishmania, but the increase was only significant in proteinuric and azotemic dogs, suggesting that uCysC would not be
63 useful to identify early kidney dysfunction in CanL (García-Martínez et al. 2015).
7.2.2. Markers of tubular damage or dysfunction
7.2.2.1.¡-glutamyl transferase (GGT)
GGT is an enzyme of the luminal side of the brush border of the proximal renal tubular cells essential in glutathione homeostasis being implied in the extracellular glutathione breakdown and as a component of cellular antioxidant defense (Hokamp and Nabity, 2016). GGT is unstable in untreated urine and their activity must be measured immediately after sampling (Paltrinieri et al. 2016)
Increase in urinary GGT:creatine ratio (uGGT/Cr) has been observed in pathological conditions leading to AKI. In dogs with pyometra, its increment was correlated with the severity of the histological lesions in the proximal renal tubules (De Loor et al. 2013). A slight to marked (2- 3 folds) increase in uGGT/Cr has been reported in gentamicin and cisplatin experimentally induced nephrotoxicosis respectively, related to tubular injury (De Loor et al. 2013).
In dogs with natural renal disease was demonstrated that uGGT/Cr could be useful to detect established AKI, but not CKD (De Loor et al.
2013). Also, uGGT values were not useful to differentiate between non- azotemic and healthy dogs (Palacio et al. 1997).
One study demonstrated that high levels of uGGT/Cr could predict the presence of proteinuria in dogs with CKD secondary to Leishmania and could be helpful to differentiate between tubular and mixed proteinuria determined by urinary SDS-AGE (Ibba et al. 2016). It has been
64 suggested that the increase in uGGT/Cr levels could be useful to evaluate renal disease progression in CanL (Ibba et al. 2016), since tubular bands in urine electroforesis probably indicate more advanced renal lesions (Zatelli et al. 2003). In contrast, another study reported that changes in uGGT/Cr were not significant neither correlated to proteinuria improvement after 1 month of treatment (Pardo-Marín et al.
2017) probably due the presence of mainly unclassified mixed proteinuria.
7.2.2.2. N-acetyl-β-D-glucosaminidase (NAG)
N-acetyl-β-D-glucosaminidase (NAG) [150 kDa] and b-glucoronidase are lysosomal enzymes of the proximal tubular renal cells (Hokamp and Nabity, 2016). It seems that uNAG is not affected by age in dogs, but in humans it is lower in children than in adults, probably due changes in muscular mass and consequently in creatinine excretion (Smets et al.
2010). NAG is unstable in untreated urine and their activity must be measured immediately after sampling (Paltrinieri et al. 2016)
Dogs with lower urinary tract infection accompanied by pyelonephritis had markedly increased uNAG:creatine ratio (uNAG/Cr) values indicating probable tubular damage. Hematuria, pyuria, and bacteriuria/lower urinary tract infection without concomitant pyelonephritis does not alter uNAG or uNAG/Cr (De Loor et al. 2013).
In humans, NAG has been described as a helpful biomarker to confirm established AKI and as a predictor of its severity and outcome (De Loor et al. 2013). The magnitude of uNAG increment has been positive correlated to lesions in proximal tubules in dogs with pyometra. A slight to marked (2-3 folds) increase in uNAG/Cr has been reported in gentamicine and cisplatin experimentally induced nephrotoxicosis,
65 respectively, related to tubular injury (De Loor et al. 2013).
Increment in uNAG/c concentration is secondary to tubular damage or increased lysosomal turnover related to an increased competition for tubular reabsorption secondary to glomerular proteinuria.
Consequently, the contribution of tubular damage to the urinary excretion of this protein could be difficult to assess (Nabity et al. 2012).
In dogs with CKD, uNAG values were significantly different compared to healthy dogs (Smets et al. 2010). In XLHN has been observed a constant increase in uNAG values during mid to late stage of the disease that could be related to a constant level of tubular damage and/or to glomerular proteinuria (Nabity et al. 2012). One study in dogs with CKD and proteinuria, shown a strong correlation with histological glomerular damage without correlation with tubulointerstitial lesions, suggesting that uNAG/Cr could also be useful to detect glomerular dysfunction in chronic proteinuric nephropathies (Hokamp et al. 2016).
Also, increased uNAG/Cr has been significantly associated with immune complex glomerulonephropathies, that in combination with uIgM/c had a moderate sensitivity of 75 and 78% respectively, to predict this group of renal diseases (Hokamp et al. 2016).
In one study, an evident significant increment of NAG and b- glucoronidase has been observed in urine of non azotemic dogs with Leishmania, compared with non azotemic healthy dogs (Palacio et al.
1997). Another study in CanL at different stages of renal disease reported an increase in uNAG levels at diagnosis, and a significant decrease in uNAG concentration correlated with improvement of proteinuria values after 1 month of treatment. In both studies, it was suggested that the increase in uNAG levels was secondary to the glomerular damage often seen in CanL, in addition to tubular damage (Pardo-Marín et al. 2017).
66 7.2.2.3. Retinol-binding Protein (RBP)
Retinol-binding protein (RBP) is 21-kDa LMW plasma protein synthesized mainly in the liver but also in the kidney, lungs, spleen, brain, stomach, heart, and skeletal muscle. It is the principal carrier of retinol (vitamin A). The RBP-retinol plasmatic complex bounds to transthyretin (TTR) that transports thyroxine and retinol. TTR-RBP complex cannot pass the glomerular barrier because its high weight.
When retinol has reached its target tissues, the affinity of RBP for TTR decreases. Free RBP can be filtered by the glomeruli and reabsorbed in the proximal tubule after its cellular catabolism (De Loor et al, 2013).
Loss of RBP in urine could be observed as consequence of decrease reabsorption of RBP secondary to tubular damage and/or competition for reabsorption in the presence of large amount of protein in glomerular damage. Loss of the TTR-RBP complex due glomerular disease could contribute to urinary RBP losses (Hokamp and Nabity, 2016).
Urinary RBP (uRBP) has been described as potential marker for proximal tubular dysfunction in humans and dogs. uRBP may be useful to predict the severity and outcome of AKI, and to predict the development of microalbuminuria and diabetic nephropathy in normoalbuminuric humans (De Loor et al. 2013).
Increased concentrations of uRBP has been observed in dogs with CKD.
Also, increment in urinary RBP:creatinine ratio (uRBP/Cr) levels has been reported in urolithiasis and XLHN by Western blot analysis, ELISA, or both in comparison with healthy dogs (Hokamp et al. 2016).
However, the utility of RBP to identify early kidney dysfunction is still
